Anti-first-pass effect compounds

ABSTRACT

Safe, effective first-pass inhibiting compounds and citrus-derived substances are provided. Formulations containing the compounds are also provided as are methods for inhibiting the first pass effect.

This application is a Continuation of U.S. application Ser. No.11/212,909, filed Aug. 29, 2005, which is a Continuation of U.S.application Ser. No. 10/647,444, filed Aug. 26, 2003, now abandoned;which is a Continuation of U.S. application Ser. No. 09/916,500, filedJul. 3, 2001, now U.S. Pat. No. 6,660,766; which is a Continuation ofU.S. application Ser. No. 09/644,860, filed Aug. 24, 2000, now U.S. Pat.No. 6,309,687; which is a Continuation of U.S. application Ser. No.09/455,911, filed Dec. 7, 1999, now U.S. Pat. No. 6,162,479; which is aContinuation of U.S. application Ser. No. 09/255,874, filed Feb. 23,1999, now U.S. Pat. No. 6,054,477; which is a Continuation of U.S.application Ser. No. 08/997,259, filed Dec. 23, 1997, now U.S. Pat. No.6,063,809; which is a 119(e) of U.S. Provisional Application Ser. No.60/056,382, filed Aug. 26, 1997.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to anti-first-pass effect compounds,compositions, and methods for their use, preparation, synthesis, andformulation. Preferably, the invention compounds and compositions areprovided as a dietary supplement or as a medical food or as some othertype of food product, or as a drug, pharmaceutical or drug preparation,or in some other physical form. In addition to any other function theyhave, the invention compounds and compositions function as inhibitors ofthe first-pass effect of orally-administered drugs. Beneficiaries ofthis invention are animals, preferably mammals, particularly humans, whorequire drugs, etc. subject to the first-pass effect.

2. Discussion of the Background

The “first-pass effect” of drugs given orally refers to the process ofdrug degradation during a drug's transition from initial ingestion tocirculation in the blood stream. Often discussed in terms of“bioavailability”, it is not uncommon for a drug that is administered toa patient orally to be given in a 5-fold or greater amount thanultimately necessary due to the degradation that occurs in the patient'sbody after intake. For example, the impact of the first-pass effect canbe demonstrated with the case of the antihistamine terfenadine, wherein99.5% of a tablet given by mouth is quickly changed to metabolites;hence, the bioavailability of terfenadine is approximately 0.5% (D.Garteiz et al., Arzneim.-Forsch., 1982; 32:1185-1190). As a furtherexample, cyclosporin A, administered to organ transplant patients, has amedian oral bioavailability of approximately 30% and a bioavailabilityrange of approximately 8-92% among patients. Because of this largeinterindividual variation in cyclosporin bioavailability, frequentmonitoring of blood concentrations during therapy initiation isnecessary.

The inhibition of a particular xenobiotic metabolism as a mechanism ofaction generally, as well as the inhibition of the first-pass effectwith chemical agents specifically, is well known in the art and has beenfor some time. Examples include the treatment of methanol (wood alcohol)poisoning with ethanol and the inhibition of the first-pass effect ofcyclosporin with ketoconazole. See, for example, First, R. M. et al.,The Lancet, 1198, Nov. 18, 1989, incorporated herein by reference.

Although the agent(s), enzyme type(s), biological processes, etc.responsible for the first-pass effect have not been fully identified,research has focused on agents capable of inhibiting the cytochrome P450system. Inhibition of the P450 system is a model for in vitrodetermination of in vivo bioavailability enhancement. See, e.g., U.S.Pat. Nos. 5,478,723 and 5,567,592, both incorporated herein byreference, for a more full description of the P450 system. As reportedby A. Keogh et al. (N. Eng. J. Med., Vol. 333, No. 10, p. 628, 1995) andS. Butman et al. (J. Heart Lung Transpl., Vol. 10, No. 3, p. 351, 1991),the dose of cyclosporin required by heart transplant patients could bereduced by approximately 85% when cyclosporin was co-administered withketoconazole. In economic terms, both references estimated the costsavings to be equal to approximately $5,000 per year per patient. Otherdrugs which are subject to the first-pass effect and whosebioavailability is increased by inhibitors commonly given to humansinclude midazolam (K. Olkkola et al, Clin. Pharmacol. Ther., 1993,53:298-305), terfenadine (Seldane®) (P. Honig et al., JAMA, Vol. 269,No. 12, 1513, 1993) and triazolam (Varhe, A. et al, Clin. Pharmocol.Ther., 1994, 56:601-7).

In addition to ketoconazole, the drugs fluconazole, ritonavir,itraconazole, miconazole, erythromycin and troleandomycin have beenidentified as inhibitors of the first-pass effect, in addition to anypharmacological effect they possess. These compounds, however, areantiviral, antimicrobial, or antifungal agents. Because of theheightened current awareness of the fact that overuse of such agents canresult in resistant microbial strains, because some of the mosteffective inhibitors are antimicrobials, and because transplant andHIV-infected patients have compromised immune systems, the use of theseinhibitors of the first-pass effect has significant drawbacks and, forexample, in the case of ketoconazole, the purposeful co-administrationof this inhibitor with drugs susceptible to the first-pass effect hasnot become widespread. In fact, the emergence of antifungal drugresistance in immunocompromised patients is already known (T. J. Walsh:“Emergence of Antifungal Drug Resistance in Immunocompromised Patients”Seminar, National Institutes of Health, Feb. 7, 1996; Georgopapadakou,N. H. et al, Antimicrobial Agents and Chemotherapy, February 1996, p.279-291).

Dietary supplements, medicines, compounds, extracts, etc. that are basedon materials isolated from nature are increasingly being studied andmade available to consumers. This trend is largely due to the fact thatobtaining patent protection for these materials has become routine (see,for example, U.S. Pat. Nos. 4,708,948, 5,409,938, 5,314,899, 5,591,770and 5,654,432, all incorporated herein by reference). Not surprisingly,this trend is now spreading to first-pass effective agents.

In 1991, Bailey et al. reported (Bailey, D. G., et al, The Lancet, Vol.337, Feb. 2, 1991, p. 268, incorporated herein by reference) thatgrapefruit juice increased the bioavailability of felodipine, andindicated that the inhibition of cytochrome P450 enzymes bybioflavonoids could explain their findings. This identification ofbioflavonoids as the active ingredient in grapefruit juice wasimmediately challenged by R. Chayen et al. (The Lancet, Vol. 337, Apr.6, 1991, p. 854) who suggested that sesquiterpenoid compounds ratherthan flavonoids were the active ingredients in grapefruit juiceresponsible for inhibition of the first-pass effect. Although Bailey andEdgar were granted a patent (U.S. Pat. No. 5,229,116, incorporatedherein by reference) directed to a method of increasing thebioavailability of a pharmaceutical agent by co-administration of aflavonoid such as naringin, their own recent work has openly broughtinto question the accuracy of their initial identification of flavonoidsas active ingredient. See, for example, Bailey et al., Clin.Pharmacokinet. 26 (2): 91-98, 1994, particularly pages 95 and 96thereof. See also Edwards, D. J. et al, Life Sciences, Vol. 59, No. 13,pp. 1025-1030, 1996.

The reported effects of grapefruit juice as an effective inhibitor ofthe first-pass effect has lead to numerous research articles regardingthe inhibition of the first-pass effect by grapefruit juice on, e.g.,nifedipine, nitrendipine, nisoldipine, cyclosporin A, midazolam,triazolam, coumarin, and caffeine. As these results have become betterknown, the so-called “grapefruit juice effect” has become the subject ofnewspaper articles, newsletters and medical texts intended for thegeneral public. See, for example, “The Medical Letter”, Vol. 37 (issue955) Aug. 18, 1995, The Peoples Pharmacy, Chapter 4 (St. Martin's Press)1996, p. 41, the Feb. 19, 1991 newspaper article regarding felodopineand grapefruit juice in the New York Times (section C, page 3, column 1)and a recent article in the Washington Post (Section A, p. 11, Aug. 30,1996).

A review of the published studies that demonstrate the grapefruit juiceeffect also shows that the magnitude of the effect varies widely, and itis the present inventors' suspicion that this variation is traceable tothe source of the juice. In fact, the production of commercial citrusjuice involves a complicated series of factors that increase thevariability of the final product's composition. These factors includethe squeezing technique, the concentration technique, the origin of thefruit, the ripeness of the fruit at harvest, the admixture of fruitsdiffering in origin and ripeness, the admixture of juice and fruittailings, etc. Because the active agents in the grapefruit juice thatinhibit the first-pass effect were unknown or misidentified, scientistsand consumers could not choose a grapefruit juice preparation and relyupon its utility to inhibit the first-pass effect.

Moreover, grapefruit juice in particular and citrus products in generalare known to contain phototoxic furocoumarin derivatives includingpsoralen, xanthotoxin and bergapten. While these compounds are usefulfor the controlled, clinical treatment of selected dermatologicaldiseases including vitiligo, psoriasis and mycosis fungoides, they arealso known to be toxic, in particular, phototoxic. Thestructure-activity relationship for the phototoxicity of furocoumarinshas been clearly delineated from human studies (for example, L. Musajoet al, Herba Hungarica, 1971, Tom. 10, No. 2-3, pp. 79-94), and thesestudies show that photosensitizing activity is removed by ringhydroxylation or by lengthening the alkyl-chain length of ethersubstituents.

Careful evaluation of the literature shows that psoralen and certain lowcarbon number ether-substituted furocoumarins that are given to humansin large doses do inhibit cytochrome P450. See, for example, D. Bickerset al., J. Investigative Dermatology, 79:201-205, 1982, M. Tinel et al.,Biochemical Pharmacology, Vol. 36, No. 6, 951-955, 1987, H.Fouin-Fortunet et al., J. Pharm. Experimental Therapeutics, Vol. 236,No. 1, 237-247, 1986, and D. Mays et al, Clin. Pharmacol. Ther.,42:621-626, 1987. Thus, and because the known successful inhibitors ofthe first-pass effect generally inhibit cytochrome P450, a temptingconclusion, particularly in view of the recent disclaimers by Bailey,and others, is that these low molecular weight furocoumarins present incitrus are the active first-pass inhibitors in grapefruit juice. Infact, and as will be described more fully below with regard to thepresent invention, the present inventor has found that this is not thecase. Because the present inventor has discovered specific compoundsthat inhibit the first-pass effect it is now possible to produce areliable, safe composition that both inhibits the first-pass effect and,if desired, that is citrus-based or of citrus origin and which containsno or reduced amounts of low molecular weight phototoxic furocoumarins.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows inhibitor results for various inhibitors.

FIG. 2 shows inhibitor results for various inhibitors.

OBJECTS OF THE PRESENT INVENTION

It is one object of this invention to provide chemical compounds andcompositions that inhibit the first-pass effect and which are in a form,concentration, purity, etc. other than that which is naturally orcommercially occurring.

Another object of the present invention is to provide a reliable, safecitrus-based or citrus-origin product that comprises one or moreinvention compounds in non-naturally and non-commercially occurringamounts and inhibits the first-pass effect and which, optionally, isfree of or contains a reduced amount (as compared to a naturally orcommercially occurring amount) of phototoxic and, optionally,non-first-pass inhibiting low molecular weight furocoumarins, which isuseful as a food or dietary supplement, a pharmaceutical, a drug, etc.

Another object of the present invention is to provide a compositioncomprising one or more invention compounds that is effective against thefirst-pass effect.

Another object of the present invention is to provide a composition thatcontains one or more of the invention compounds and no or reducedamounts as compared to naturally or commercially occurring amounts ofphototoxic low molecular weight furocoumarins.

Another object of the present invention is to provide a compositioncomprising at least one invention compound and providing consistent andreliable first-pass inhibiting activity.

Another object of the present invention is to provide theabove-described compounds and compositions as a component of productsand mixtures that provide active ingredients, therapeutic agents, drugs,etc. or other substances that are subject to the first-pass effect inhumans.

Another object of the present invention is to provide first-pass effectinhibiting compounds, also called bioenhancers and inhibitors herein, innon-natural and non-commercially occurring forms.

Another object of the present invention is to provide mixtures of one ormore invention first-pass effect inhibiting compounds with varioustherapeutic agents, active agents, drugs or other substances(hereinafter referred to as “drugs”) that are subject to the first-passeffect.

Another object of the present invention is a method for inhibiting thefirst-pass effect in human patients, animals, etc. taking drugs having afirst-pass effect.

Another object of the present invention is a method for preparing theabove-described compositions, compounds, mixtures, etc.

Another object of the present invention is a method for preparing acitrus-based or citrus-origin composition containing no or reducedamounts as compared to naturally and commercially occurring amounts ofphototoxic and non-first-pass inhibiting furocoumarins preferably usingreagents that the U.S. Food and Drug Administration regards may be usedfor food or drug manufacturing, including GRAS materials (in thisapplication, “non-first-pass inhibiting” includes first-pass activityprovided by 2000 nM bergamottin or imperatorin according to Protocol Cor C′ herein).

Another object of the present invention is to provide and use first-passeffective compounds and compositions containing a first pass effectiveamount (in aggregate or individually) of at least one invention compoundin isolated form and/or pyrogen-free form and/or sterile form and/orsubstantially pure form and/or pharmaceutical form and/or chemicallypure form and/or in a form comprising a higher concentration or purityof invention compounds than found both in nature and commercially. Asused herein “commercially” means products produced and sold locally,nationally and internationally, especially by the citrus-processingindustry. These forms, as their names specify, are different from theinvention compounds, as they naturally occur in, for example, commercialcitrus and in citrus products such as juice, cold pressed oils, juiceconcentrates, oils, etc.

Another object of the invention is to provide a method of inhibiting thefirst-pass effect by administration of at least one invention compound,composition etc. to humans.

These and other objects will become apparent to those of ordinary skillin this art upon a full appreciation of the invention as described belowwith regard to preferred embodiments.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present inventor has discovered chemical compounds which inhibit thefirst-pass effect of orally administered drugs in humans. The presentinventor has also discovered that phototoxic low molecular weightfurocoumarins and certain ether-substituted furocoumarins that arenaturally present in citrus extracts, juices, byproducts, etc. may beremoved therefrom or reduced in concentration without destroying thefirst-pass effect inhibiting compounds therein. The present inventor hasalso discovered a method for preparing citrus-based compositions usingonly FDA or USP acceptable reagents. The present invention has beencompleted on the basis of these findings and will be described in moredetail below.

The invention chemical compounds which inhibit the first-pass effect inanimals, including humans, are, in one preferred embodiment, compoundsaccording to the following Formulae I-IV:

In each of the above structures, R is, independently, H or an optionallysubstituted C₁-C₁₅ alkyl group,

L is an optionally substituted C₁-C₁₅ linear or branched, saturated,monounsaturated or polyunsaturated alkyl group optionally interrupted byone or plural nonadjacent sulfur or oxygen atoms and optionallyterminated at one or both ends by oxygen,

HAr is an optionally substituted C₆-C₂₄ aromatic group or heteroaromaticgroup optionally containing one or plural ring atoms selected from thegroup consisting of N, O, S, and P,

and E is —OH, —COOH, —COOR (where R is defined above) or an optionallysubstituted C₁-C₈ linear or branched, saturated, monounsaturated orpolyunsaturated alkyl group optionally interrupted by one or pluralnonadjacent oxygen or sulfur atoms, or E is a C₃-C₈ optionallysubstituted cyclic saturated, monounsaturated or polyunsaturated alkylgroup optionally interrupted by one or plural nonadjacent oxygen orsulfur atoms, or E is optionally substituted HAr. Preferably, thecompounds of Formulae I-IV as well as those described below do notcontain a peroxide (0-0) group. Disulfide groups (S-S) are notpreferred, but may be present. Preferably E is an epoxide or dihydroxyradical such as —CH(OH)²⁻. E may also be an acid-opened epoxide group.

The compounds of the invention as described above are unlimited withregard to stereocheraistry, E-Z isomerism and all possibilities areincluded. Racemic mixtures are included as are each and every enantiomerand diasteriomer. Preferred stereochemistry is shown later.

The groups R, L, HAr, and E may optionally be substituted with a C₁-C₆linear, branched or cyclic alkyl group, —OH, a halogen atom, a C₁-C₅alkoxy group, a C₁-C₅ alkyl carbonyloxy group, a C₁-C₅ alkoxycarbonylgroup, etc. Such substituents also may be optionally substituteddirectly on the ring structures of Formulae I-IV regardless of whethersuch substituents appear on R, L, HAr or E.

A second preferred embodiment of the present invention chemicalcompounds which inhibit the first-pass effect are depicted by FormulaeV-X:

As noted above for Formulae I-IV, Formulae V-X are unlimited with regardto stereochemistry, E-Z isomerism, etc.

The most preferred compounds according to the present invention, whichinhibit the first-pass effect, are those according to the secondembodiment above and having the following stereochemistry (formulaeXI-XVI):

The compositions of the present invention contain at least one inventionfirst-pass effective chemical compound preferably in a first-passeffective amount. Citrus-based compositions of the invention furthercontain a citrus-derived extract, concentrate, peel, juice, oil,by-product, etc., (hereinafter referred to as the citrus-derivedsubstance) and may be provided by any combination of these forms and maybe derived from more than one citrus fruit. Useful citrus fruits hereininclude grapefruit, lemon, lime and, preferably, any citrus fruitnaturally containing an invention first-pass effect inhibiting compoundor mixture of such compounds. Prior work in the field indicates that acommon type of orange (Citrus sinensis) does not inhibit the first-passeffect. Citrus fruits that contain one or more substances that inhibitthe first-pass effect are included in the invention, including all crossbreeds, etc. and are referred to herein as “first-pass citrus”. Apreferred citrus fruit useful in the present invention is grapefruit.

First-pass effective compounds, substances and compositions describedherein are materials that prevent or retard the degradation of orallyadministered drugs in the body. Preferably, the first-pass effectivematerials of the invention, including substances, compositions,mixtures, invention compounds, etc. increase drug bioavailability by atleast 1%, preferably by more than 5% and most preferably by more than15% including 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 100, 150, 200, 250, 300, etc. percent as measured by the Area Underthe Curve (AUC) method. See U.S. Pat. No. 5,567,592 incorporated hereinby reference. A several-fold, including 5, 10, 15, 20-fold, etc.increase in bioavailability (i.e., several hundreds or thousands ofpercent AUC increase) is not unusual with the present invention. Thefirst-pass effectiveness of invention compounds, composition, mixtures,materials, etc. may also be measured by, and preferably meet thecriteria of, the methods and characterizations described in WO 97/15269,U.S. Pat. No. 5,665,386 and PCT/US96/09607, all incorporated herein byreference.

Preferred citrus-derived substances of the invention includecold-pressed citrus oil, particularly cold-pressed grapefruit, lime,lemon, etc., oil, and citrus by-products including tailings from citruspacking/juice plants. Cold-pressed citrus oils, including cold-pressedorange (except Citrus sinensis), grapefruit, lime and lemon oil, arecommodities and are described, for example, in the Food Chemicals Codex,Fourth Edition, National Academy Press, Washington, D.C. 1996,incorporated herein by reference. Other citrus-derived substances usefulherein include the various other citrus oils (distilled, essential,desert type, etc.), bitter cold-pressed oils, etc. Geographical originof the invention citrus providing the citrus-derived substance isunimportant herein. Citrus juices or peel (rind) may also be used, aswell as any first-pass effective solid, semi-solid or liquid portion ofa first-pass citrus. Mixtures may be used.

The citrus-derived substance present in invention compositions may makeup the entire citrus-based composition or may be only a part thereof.Thus, if the citrus-based substance is prepared such that it containsone or more compounds according to Formulae I-XVI in a first-passeffective amount no further compound need be added. Food grade orpharmaceutically acceptable diluents, excipients, carriers, etc., may beadded, if desired.

The citrus-derived substance of the present invention composition ispreferably treated so as to reduce the amount of phototoxic and,optionally, non-first-pass effective, furocoumarins naturally presenttherein. Preferably, these furocoumarins are completely removed, meaningthat they are removed to an extent such that their presence isundetectable by liquid and, preferably, gas chromatography.

The invention method for removing phototoxic low molecular weightfurocoumarins from invention citrus-derived components preferablycomprises optional removal of volatile components (components removedafter 12-48 h at a pressure of 10⁻²-10⁻³ Torr) and extraction withmixtures of at least one C₁-C₁₀ alcohol (preferably ethanol) and water,optionally in the presence of base. In certain situations it ispreferable not to remove volatile components such as naturally-occurringterpenes but rather to use these volatiles essentially as solvent infurther processing. The extraction mixture of alcohol and water may bediscarded and what is left is useful herein. C₂-C₅ alcohols are alsopreferred as are C₂ and C₃ and C₄ alcohols. The alcohol (ethanol) mayeither be 100% alcohol or may be conveniently supplied and used incommonly available alcohol-water dilutions (e.g., 95% ethanol/5% water,etc.). In all cases the alcohol (ethanol) reagent is preferably U.S.P.grade or better. The water used herein for extracting the inventioncitrus-derived substance (component) is preferably distilled water, andis also preferably U.S.P. grade or better. Any combination of solventsor single solvent may be used herein for extraction. The solvent(s) arepreferably FDA acceptable for food and drug manufacturing.

The present invention method for removing phototoxic low molecularweight furocoumarins may include successive extractions with alcohol(ethanol)/water mixtures, and the successive alcohol (ethanol)/watermixtures used may either be of the same volume ratio or different volumeratios. Preferred alcohol (ethanol):water volume ratios range from1:10-10:1, are more preferably 1:1 (±3%, 5%, 8% or 10%) and may be 20-80or 45-60% alcohol (ethanol) on a volume/volume basis, and include 2:1,3:1, 1:2, 1:3, etc. as well as 55/45, 60/40, 65/35, 70/30, 10/90, 15/85,20/80, 25/75, 30/70,35/65, 40/60, 45/55, 40/60, 35/65, 30/70, etc.alcohol/water. The extractions may be accomplished by any method knownin the art including liquid-liquid extraction, liquid-solid extraction,continuous extraction etc. When the raw material used to prepare theinvention citrus-derived extract is, for example, an oil, the alcohol(ethanol)/water mixture used for extraction can be simply added thereto,shaken therewith, and separated naturally or with the help of acentrifuge. Repeated extraction is helpful, as are continuous extractionmethods such as countercurrent extraction, etc.

As noted above, base is preferably used in removing phototoxicfurocoumarins and may be added to the water or alcohol or both.Preferred bases are the alkali and alkaline earth hydroxides and oxides,most preferably sodium hydroxide and potassium hydroxide. The base isgenerally present in amounts from 0.01-80 grams per liter ofalcohol/water mixture.

Preferably, the invention method for removing phototoxic low molecularweight furocoumarins significantly diminishes, and preferably completelyremoves beyond the detection limits of liquid and, preferably, gaschromatography, methoxy-substituted linear and angular furocoumarinsincluding xanthotoxin (8-methoxypsoralen), bergapten(5-methoxypsoralen), isobergapten, isopimpinellin, etc., andunsubstituted linear and angular furocoumarins (psoralen, angelicin,etc.). Furocoumarins that have been determined herein to be ineffectivefirst-pass effect furocoumarins may also be removed, if desired. Thesecompounds include bergamottin, psoralen, angelicin, isopimpinellin,marmin, 6′,7′-dihydroxybergamottin, and imperatorin.

The invention citrus-derived substance, invention compositions,invention mixtures, invention pharmaceutical compositions, etc.preferably contain a first-pass effective amount of at least onefirst-pass effective compound of Formulae I-XVI above. In thealternative, several compounds of Formulae I-XVI may be present, each innon-first-pass effective amounts where the sum of the concentrations ofsaid compounds provides first-pass effectiveness.

In addition to the description above, one or more of the hydrogen atomsdepicted in these formulae (i.e., Formulae I-XVI) may be replaced by oneor any combination of two or more of hydroxy, halogen, linear orbranched C₁-C₄₀ hydrocarbon, C₁-C₄₀ linear or branched ether (—OR whereR is linear or branched hydrocarbon), C₁-C₄₀ alkylhydroxy (—ROH where Ris linear or branched hydrocarbon and OH is bonded to a primary,secondary or tertiary carbon), etc. As used herein “hydrocarbon” meansbranched and linear alkyl and branched and linear alkenyl. Alkenyl isany hydrocarbon with at least one double bond but including multipleconjugated and nonconjugated double bonds. All salts, particularlypharmaceutically acceptable salts, and stereoisomers, physical forms,etc. are also included. The compounds described in formulae I-XVI may besynthesized by any general technique known in the art, and theirsynthesis is within the skill of the ordinary artisan in this field. Nowthat they have been identified they can also be isolated from acitrus-derived substance as shown herein.

Preferred methods for making the invention compounds of Formulae I-XVIinclude the following schemes:

Such reactions are within the skill of the ordinary artisan. See, forexample, Chemistry Letters, 2019-2022,1990, Can. J. Chem., 63:2673-2678, 1985, Australian Journal of Chemistry, 42: 1235-1248, 1989,East German patent DD 275687 and Soviet Union patent SU 1397449, allincorporated herein by reference. Although each of the reaction schemashown above yield either Formulae I+II or Formulae III+IV, each methodcan give Formulae I-IV if the appropriate reactants are used. Thepresent invention compounds, citrus-derived substance, mixtures,pharmaceutical compositions, etc. preferably contain at least onecompound according to Formulae I-XVI above. Mixtures may be used.

The use of present invention citrus-derived substance, compositions,mixtures, inhibitors, compounds, etc. are not limited and may preferablybe administered in amounts of 2 nanograms—2 g and more per patient perday to increase the bioavailability of drugs taken orally by a patient.Compositions of the invention may contain, preferably, more of theinvention compounds than naturally present in citrus products. Dosagesare determinable by those of ordinary skill in the art and depend uponthe extent to which a, e.g., active agent (drug) is subject to thefirst-pass effect, etc. Dosage forms include oral administration forms,topical administration forms, injection forms. The invention compounds,citrus-derived substance, compositions, mixtures, etc. may optionally bepart of or added to a citrus-based composition or other edible materialwhich is preferably a taste-masking flavor, juice, etc.

The citrus-derived substance, mixtures, compositions and compounds ofthe invention inhibit the first-pass effect of drugs taken orally byhumans and other animals. A “first-pass effective amount” of aninvention material is any amount that increases the oral bioavailabilityof any substance by any amount (e.g., 1%, 5%, 10%, etc.; see above wherethe AUC method is described, including all values and ranges betweenthese values) as compared to the case where no invention material isadministered in such a situation. A “first-pass effective” inventioncitrus-derived substance, mixture, composition or compound is a materialthat inhibits the observed first-pass effect of at least one drug in ananimal, preferably a human, preferably the first-pass effect caused bythe cytochrome P450 system. This is also referred to herein asanti-first-pass activity. Administration is preferablyco-administration, meaning just before, just after, or with drug, activeagent, therapeutic agent, medical food, etc. subject to the first-passeffect. “Just before” and “just after” include all times where theinvention material provides a benefit by inhibiting the first-passeffect. Preferred forms of the invention comprise the inventioncompounds, citrus-derived substance, mixture, composition, etc. insideof, e.g., a gel capsule, or co-formulated with food-grade orpharmaceutically-acceptable binders, diluents, etc. Dosage forms (saltor base, tablet or gum, etc.) as well as binders, salt forms,excipients, etc. which are useful are found in, e.g., U.S. Pat. Nos.5,576,448, 5,576,446, 5,576,437, 5,576,439, 5,576,438, 5,576,337,5,576,339 and 5,576,336, all incorporated herein by reference. Theinvention citrus-derived substance, mixtures, compositions and compoundsare preferably provided in an amount that provides consistent, reliablepotency from batch to batch regardless of the form in which it isprovided.

The word “drug” as used herein is defined as a chemical capable ofadministration to an organism which modifies or alters the organism'sphysiology. More preferably the word “drug” as used herein is defined asany substance intended for use in the treatment or prevention ofdisease, particularly for humans. Drug includes synthetic and naturallyoccurring toxins and bioaffecting substances as well as recognizedpharmaceuticals, such as those listed in Merck Index, Twelfth Ed., MerckResearch Laboratories, Whitehouse Station, N.J., 1996, “The PhysiciansDesk Reference,” 47th edition, 1993, pages 101-321; “Goodman andGilman's The Pharmacological Basis of Therapeutics” 8th Edition (1990),pages 84-1614 and 1655-1715; and “The United States Pharmacopeia, TheNational Formulary”, USP XXII NF XVII (1990), the compounds of thesereferences being herein incorporated by reference. The term drug alsoincludes compounds that have the indicated properties that are not yetdiscovered or available in the U.S. The term drug includes pro-active,activated and metabolized forms of drugs. The present invention can beused with drugs consisting of charged, uncharged, hydrophilic,zwitter-ionic, or hydrophobic species, as well as any combination ofthese physical characteristics. A hydrophobic drug is defined as a drugwhich in its non-ionized form is more soluble in lipid or fat than inwater. Preferably, a hydrophobic drug is defined as a drug more solublein octanol than in water. See U.S. Pat. No. 5,567,592, incorporatedherein by reference. The invention can be used with humans and animalssuch as mammals.

The present invention compounds and citrus-derived substances may beco-formulated with drugs, preferably drugs that are subject to thefirst-pass effect. Preferably the drug has an oral bioavailability of92% or less, more preferably 50% or less. Examples include, in additionto those incorporated by reference above, saquinavir, indinavir,L-deprenyl, tacrolimus, cyclosporin A (Sandimmune®), cyclosporin A(Neoral®), nelfinavir, VX-478/141 W94, felodipine, nifedipine andsumatriptan. Such co-formulations include the invention citrus-derivedsubstance and/or one or more compounds in amounts mentioned above with,typically, lesser amounts than currently necessary of drug activeingredients that are subject to the first-pass effect. Binders,diluents, etc. acceptable for pharmaceutical use can also be added. Oneof ordinary skill in the art is capable of determining the dosage of theinvention compounds based on simple testing procedures well known in theart and including pharmacological experiments which determine the amountof drug in the blood stream over a given time period afteradministration.

Other products useful for co-formulation herein are any and all drug,medical food, or other products that are subject to the first-passeffect. Examples of drugs are listed in the Merck Index, Twelfth Ed.,Merck Research Laboratories, Whitehouse Station, N.J., 1996,incorporated herein by reference. Determining whether a substance issubject to the first-pass effect is within the skill of the averageartisan in this field.

It is preferred that invention materials be protected from stomach acidby, e.g., a coating. Such coatings are well known in the art, andinclude enteric coatings, etc. See the Kirk-Othmer Encyclopedia ofChemical Technology, 3rd Ed. Vol. 17, p. 281 ff, incorporated herein byreference. Other useful pharmaceutical forms may also be used, such astime-release forms (coatings), hard- and soft-shell gelatin capsules,etc.

EXAMPLES Synthesis of a Spiro Ortho Ester (Formula XVII)

Benzyl 6,7-epoxygeranyl ether was placed in an evacuated chamber (0.1torr) overnight in order to remove extraneous water from the sample; 190mg (730 μmol) was weighed. Three equivalents of 7-methoxycoumarin (0.386g) was dissolved in 3 mL CH₂Cl₂, and this liquid was transferred to aclosed glass container. Helium was used to purge the system during thereaction period, the container was maintained at 5-6° C., and thereaction solution was magnetically stirred. Forty-three μL of a 1 Msolution of SnCl₄ in CH₂Cl₂ and 16 μmol of BF₃•Et₂O were added; 5minutes passed, then the epoxide, dissolved in 4 mL CH₂Cl₂, was added in4 equal portions; each portion was followed by 4 μmol BF₃•Et₂O. Thereaction was held at 5-6° C. and stirred for 3.5 hours, and the reactionapparatus was then disassembled. The reaction mixture was treatedimmediately as follows: the mixture contains a total of 75 μmol of BF₃and SnCl₄, so 3 equivalents of pyridine (225 μmol) were added (reactionmixture held at 5-6° C., with stirring) to destroy the Lewis acidcatalysts. After 30 minutes of stirring, the solvent was removed, andthe residue was dissolved in 3.16 mL of 95% ethanol; 0.6 mL of 50% KOHin water (w/v) was added, and the solution was mixed vigorously. Water(2.24 mL) was added, and this solution was then placed in a Speed Vacapparatus overnight to remove the ethanol. Approximately 50% of theoriginal volume remained. The aqueous solution was extracted twice with3 mL CH₂Cl₂, and the pooled CH₂Cl₂ was extracted twice with 10 mL 5% KOHin water (w/v) and twice with 10 mL 5% NaCl in water (w/v). The CH₂Cl₂was removed, and the residue was dissolved in acetonitrile.

This acetonitrile solution may be used directly for HPLC purification ofthe spiro ortho ester product via the preferred conditions that follow.Linear gradients were used for elution and were formed by mixing mobilephase A composed of water with mobile phase B composed of acetonitrile(instrument: Hewlett Packard). The elution time, in minutes, as well asthe percentage of acetonitrile present in the mixed mobile phase were asfollows: 0, 55; 5, 55; 10, 90; 11, 98; 17, 98; 18, 55; 22, 55. Thechromatographic column had dimensions of 250 mm length×4.6 mm internaldiameter, was packed with C18 bonded to 4 micron silica particles (9%carbon load; YMC, Inc.), was protected with a 23 mm length×4 mm internaldiameter column containing the same material and with a 0.5 micronfilter, and was maintained at 40±0.2° C. The flow rate was maintained at1.0 mL/min during the 22 min run cycle. The column eluate from each 10μL injection was monitored at 259±2 and was fractionated using a roboticcollector (Gilson). The retention time of Formula XVII in this systemwas 13.4 minutes. ¹H N.M.R. δ (400 MHZ, CD₃OD) 1.15, s and 1.26, s and1.39, s, 4′-Me (diasteriomers); 1.64, s, 8′-Me; 1.6-1.9, m, H6′,6′;2.0-2.3, m, H7′; 3.70, s, 7-OMe; 3.83, m and 4.20, t, H5′(diasteriomers); 4.01, d, H10′; 4.45, s, H12′; 5.39, t, H9′; 5.45, d,H3; 6.40, s, H8; 6.49, d, H6; 6.71, d, H4; 7.06, d, H5; 7.20-7.35, m,phenyl. Mass spectrum m/z (electrospray): MS, 437 (MH⁺); MS/MS. 261,177, 153 (fragments of MH⁺).

Bulk Pretreatment of Cold-Pressed Grapefruit Oil for SubsequentFractionation by Reversed-Phase HPLC

1. Prepare 1:1 ethanol:water (v/v) containing 12.5 g KOH/L usingdenatured reagent alcohol.

2. Mix 1.0 L of cold-pressed grapefruit oil and 330 mL of the basicethanolic solution in a 2 L separatory funnel for 2.0 min.

3. Wait 5.0 min, then remove the bottom ethanolic phase from the funnel.

4. Repeat steps 2 & 3 four times using fresh 330-mL portions of thebasic ethanolic solution.

5. Repeat steps 2 & 3 once using a 330-mL portion of: 1:1 ethanol:waterthat is prepared without KOH. When KOH is absent, a several-hours waitis required (preferably overnight) in order for the phases to clearlyseparate. Assessment of the bottom ethanolic phase with pH paper shouldshow that the solution pH is near neutral (≅6, in most cases).

6. Place the oil phase (yield should be greater than 0.9 L) in a vacuumchamber and place the system under vacuum (0.5 torr or better) for 3-4days. The process is complete when swirling the viscous liquid whileunder vacuum no longer initiates boiling.

7. Wash the nonvolatile material with portions of acetonitrile, to atotal of 200 mL, and separate the acetonitrile-soluble and -insolublematerials by centrifugation (5 min at speed 50, IEC model K2).

8. Remove the acetonitrile from the acetonitrile-soluble phase (step 7)using a Speed Vac apparatus, and weigh the residue (22-25 g would beexpected).

9. Add acetonitrile to the residue such that each 5-mL portion contains1.5 grams of residue, and divide the solution into 5-mL portions.

10. Add 15-mL of iso-octane to each 5-mL portion, cap, vortex mix, andcentrifuge the mixture (2 min at speed 35). Discard the top iso-octanephase.

11. Repeat step 10 nine times; acetonitrile should be added occasionallyto insure that the bottom phase volume approximates 5 mL.

12. Remove the acetonitrile from the bottom phase (step 11) using aSpeed Vac apparatus, and weigh the residue (approximately 20% of theoriginal weight {step 8} would be expected).

13. Dissolve the residue (step 12) in acetonitrile such that a 0.25-0.30g/mL solution is established, filter the solution through a 0.2 μmTeflon cartridge, and store the solution at −20° C.

Bulk Pretreatment of Cold-Pressed Grapefruit Oil for Further Use asDietary Food Supplement, Drug, conFormulation Ingredient, etc.

1. Prepare a 70:30 water:ethanol solution (v/v) that contains 5%potassium hydroxide (w/v) using USP-grade ethanol, NF/FCC-grade KOH, andpurified water.

2. Mix the ethanolic solution prepared in step 1 with an equivalentvolume (or slight excess) of whole, untreated cold-pressed grapefruitoil (Food Chemicals Codex grade), and transfer the mixture to a heat-and pressure-resistant food-grade container.

3. The sealed container is maintained at 95-100° C. for 1 hour. Thecontainer is cooled, the ethanolic phase (lower phase of two-phasesystem) is removed, and a fresh portion of ethanolic solution(equivalent to the volume used in step 2) is added.

4. Repeat the boiling cycle (step 3) until the desired degree of samplepurity is achieved. Ten cycles will remove >99% of polar coumarins andfurocoumarins, will remove >90% of prominent nonpolar coumarins andfurocoumarins (i.e., epoxyaurapten, epoxybergamottin), and will notappreciably decrease the content of the inhibitory Spiro ortho esters.

5. Wash the oil with purified water until the discarded wash water pHbecomes neutral.

6. Place the oil phase under vacuum (0.1-0.3 torr) until volatilematerials are no longer removed from the sample (as assessed by, forexample, inspection of an empty in-line trap maintained at −60 to −90°C.). In general, approximately 95% of the volume of the sample will beremoved in this step.

7. Mix the product of step 6 with an equivalent volume of USP ethanol,and centrifuge the mixture. Repeat until the bottom phase issubstantially free of Spiro ortho ester inhibitors. This method removesethanol-insoluble materials from the oil novolatile preparation.

8. Optionally, but preferably, place the pooled ethanol extracts fromstep 7 under vacuum until the ethanol has been substantially removed(e.g., 99%) or reduced (e.g. 10%).

Because adulteration of raw materials is known in the food, flavor, andfragrance industries, citrus-derived components of the inventionincluding cold-pressed citrus oils should preferably be assessed beforethey are used further in the production of, e.g., compositions ofdietary supplements containing a first-pass effective amount of one or amixture of compounds of Formulae I-XVI. One strategy consists of samplepreparation (Protocol A; Protocol A′), followed by chromatography(Protocol B; Protocol B′; Protocol B″), and ending with comparisons tohistorical standards. Such assessment can provide consistent batches.

The following protocols are useful in preparing various embodiments ofthe invention.

Protocol A: Preparation of Citrus Oils for Further ChromatographicProcessing or for Administration to Humans by Removal of Toxic, LowMolecular Weight Furocoumarins.

A volume of cold-pressed citrus oil (Food Chemicals Codex grade) wastransferred to a container, and all volatile materials were removed.Although several methods exist for removing volatiles (e.g.,distillation, distillation under reduced pressure, evaporation underambient conditions), the preferred method uses Speed Vac concentrators(Savant Instruments; process requires 12-24 h and pressures of 10⁻²-10⁻³torr, and the system is run without added heat) because this method isgentle and expedient. The nonvolatile product yield is generally 0.04 to0.1 times the initial volume and is a viscous liquid.

Low molecular weight, phototoxic furocoumarins were removed from thenonvolatile preparation by liquid-liquid extraction: 16 times the volumeof viscous liquid of 1:1 ethanol:water (v/v; each U.S.P. grade) wereadded to the nonvolatile preparation, the container capped, the solutionmixed vigorously, the container centrifuged (International EquipmentCompany, Model K-2, 5 min at setting 35), and the top ethanolic phasediscarded. The extraction was repeated twice. Extraneous water andethanol may be removed from the preparation if desired by use, e.g., ofa Speed Vac apparatus. The product of this process may be used for humanadministration in, e.g., filled capsules.

Protocol A′: Pretreatment of Citrus Oils Prior to Chromatography.

Most citrus oils are not directly suitable for long-term preparativehigh pressure liquid chromatography because of the substantial presenceof materials that show poor solubility in the preferred mobile phasesystems. Hence the sample preparation protocol that follows is usedprior to chromatography.

Cold-pressed citrus oil (Food Chemicals Codex grade) is transferred to asuitable container, and all volatile materials are removed under reducedpressure (10⁻²-10⁻³ torr, 3-4 days). The nonvolatile product yield isgenerally only 5-10% of the original volume. The citrus nonvolatiles aremixed with acetonitrile in a ratio of 2:1 (w/w), the mixture iscentrifuged (International Equipment Company, Model K-2, 5 min atsetting 35), and the upper acetonitrile-containing phase is removed. Theextraction with acetonitrile is repeated once, the lower phase isdiscarded, the first and second acetonitrile phases are pooled, andacetonitrile is removed using Speed Vac concentrators (SavantInstruments; 12 h at 10⁻²-10⁻³ torr, without added heat). Thenonvolatile material is mixed with ethanolic base (1:1 ethanol:water{v/v; each U.S.P. grade} containing 12.5 g potassium hydroxide/L) in aratio of 1:4 (w/v), the mixture is centrifuged for 5 min at setting 35,and the upper ethanolic phase is removed and discarded. The nonvolatilematerial is washed an additional nine times with ethanolic base and,then, once with 1:1 ethanol:water (v/v). The residue that remains isextracted twice with sufficient volumes of acetonitrile such that allcolored material is removed. The acetonitrile solution is washed sixtimes with two volumes of hexane or iso-octane, with each hexane extract(upper layer) being removed and discarded, and the resultingacetonitrile solution is filtered through a 0.2 micron Teflon® membraneand evaporated to dryness using a Speed Vac concentrator.

The final product of the above process should appear as a viscous, deepred oil, but seasonal variations in the starting material (citrus oils)apparently can change the quality and appearance of the product of theabove process. Hence, if a copious orange crystalline materialcontaminates the deep red oil, then the number of additional washes withethanolic base should be increased from nine to nineteen.

Protocol B: Chromatography Methods for Processed Citrus Oils

The product of the above Protocol A is not suitable for any highpressure liquid chromatography because of the substantial presence ofmaterials that are not soluble in the preferred mobile phase systems.Hence the sample preparation protocol that follows is used prior tochromatography. One volume of the product of Protocol A is mixed withfour volumes of acetonitrile, the container is capped, the solution ismixed vigorously, the container is centrifuged (5 min at setting 35),and the top acetonitrile layer is filtered through a 0.22 micron Teflon®membrane. The filtered solution is stored in a closed container at −20°C. for 2 days or more and then is passed through filter paper while coldto remove a copious precipitate. The precipitation and filtration stepis repeated once. The volume of the acetonitrile solution is noted, andthe acetonitrile is removed using a Speed Vac apparatus. The residue isdissolved in half the original volume of acetonitrile, taking care notto disturb any crystalline precipitate, and the solution may now be usedfor HPLC assessment.

If preparative fractionation of the washed nonvolatile portion of citrusoil is desired, then the HPLC conditions given below are preferred.Linear gradients are used for elution and are formed by mixing mobilephase A composed of water with mobile phase B composed of acetonitrile(instrument: Hewlett Packard). The elution time, in minutes, as well asthe percentage of acetonitrile present in the mixed mobile phase are asfollows: 0, 75; 5, 75; 10, 90; 11, 98; 17, 98; 18, 75; 22, 75. Thechromatographic column has dimensions of 250 mm length×4.6 mm internaldiameter, is packed with C18 bonded to 4 micron silica particles (9%carbon load; ODS-L80, YMC, Inc.), is protected with a 23 mm length×4 mminternal diameter column containing the same material and with a 0.5micron filter, and is maintained at 40+/−0.2° C. The flow rate ismaintained at 1.0 mL/min during the 22 min run cycle. The column eluatefrom each 25 uL injection is monitored at 400+/−200 nm and at 310+/−2 nmand is fractionated using a robotic collector (Gilson).

If qualitative or quantitative assessments of citrus oils, fractionsthereof, or reference standards are desired, then the HPLC conditionsgiven below are preferred. Linear gradients are used for elution and areformed by mixing mobile phase A composed of water with mobile phase Bcomposed of acetonitrile (instrument: Hewlett Packard). The elutiontime, in minutes, as well as the percentage of acetonitrile present inthe mixed mobile phase are as follows: 0, 10; 5, 10; 30, 80; 40, 80; 41,95; 50, 95; 53, 10; 60, 10. The chromatographic column has dimensions of150 mm length×2.0 mm internal diameter, is packed with C18 bonded to 4micron silica particles (14% carbon load; ODS-M80, YMC, Inc.), isprotected with a 2 mm internal diameter column packed with a proprietarymaterial (Prism, Keystone Scientific, Inc.) and with a PTFE filter, andis maintained at 35+/−0.2° C. The flow rate is maintained at 0.20 mL/minduring the 60 min run cycle. The column eluate from each 10 uL injectionis monitored for absorbance at 400+/−200 nm and at 310+/−2 nm and forfluorescence with excitation at 229 nm, emission at 450 nm, and bandpassfiltration at 370 nm.

Protocol B′: Chromatography Methods for Processed Citrus Oils

If preparative fractionation of the washed nonvolatile portion of citrusoil (product of Protocol A′) is desired, then the HPLC conditions givenbelow are preferred. Linear gradients are used for elution and areformed by mixing mobile phase A composed of water with mobile phase Bcomposed of acetonitrile (instrument: Hewlett Packard). The elutiontime, in minutes, as well as the percentage of acetonitrile present inthe mixed mobile phase are as follows: 0, 75; 5, 75; 10, 90; 11, 98; 17,98; 18, 75; 22, 75. The chromatographic column has dimensions of 250 mmlength×4.6 mm internal diameter, is packed with C18 bonded to 4 micronsilica particles (9% carbon load; ODS-L80 YMC, Inc.), is protected witha 23 mm length×4 mm internal diameter column containing the samematerial and with a 0.5 micron filter, and is maintained at 40+/−0.2° C.The flow rate is maintained at 1.0 mL/min during the 22 min run cycle.The column eluate from each 25 uL injection of acetonitrile solutionobtained by Protocol A′ is monitored at 400+/−200 nm and at 310+/−2 nmand is fractionated using a robotic collector (Gilson).

If qualitative or quantitative assessments of citrus oils, fractionsthereof, or reference standards are desired, then the HPLC conditionsgiven below are preferred. Linear gradients are used for elution and areformed by mixing mobile phase A composed of water with mobile phase Bcomposed of acetonitrile (instrument: Hewlett Packard). The elutiontime, in minutes, as well as the percentage of acetonitrile present inthe mixed mobile phase are as follows: 0, 10; 5, 10; 30, 80; 40, 80; 41,95; 50, 95; 53, 10; 60, 10. The chromatographic column has dimensions of150 mm length×2.0 mm internal diameter, is packed with C18 bonded to 4micron silica particles (14% carbon load; ODS-M80, YMC, Inc.), isprotected with a 2 mm internal diameter guard column packed with aproprietary material (Prism, Keystone Scientific, Inc.) and with a PTFEfilter, and is maintained at 35+/−0.2 C. The flow rate is maintained at0.20 mL/min during the 60 min run cycle. The column eluate from each 10uL injection is monitored for absorbance at 400+/−200 nm and at 310+/−2nm and for fluorescence with excitation at 229 nm, emission at 450 nm,and bandpass filtration at 370 nm.

Protocol B″: Purification of Invention Compounds using a Chiral HPLCColumn

Pooled residues that result from fractionation of the 11-12.5 min region(Protocol B or B′) and solvent removal (Speed Vac, no heat added) aresubjected to chiral liquid chromatography. Isocratic elution is employed(mobile phase consists of 3.4 L iso-octane, 0.6 L 95% ethanol {remainderis water}, and 0.2 L isopropanol) to elute Inhibitors XI-XVI from thecolumn (250×4.6 mm, Keystone Scientific, Inc., Chiral from each 25 uLinjection (residue is dissolved in mobile phase) is monitored at400+/−200 nm and at 310+/−2 nm and is fractionated using a roboticcollector (Gilson).

Protocol C: Assessment of Human Cytochrome P450-MediatedBiotransformation

The process of preparing incubation mixtures begins by mixing 10 uL ofethanol or an ethanolic solution containing an inhibitor with 100 uL of100 mg/mL bovine serum albumin (Sigma) dissolved in reaction buffer atroom temperature. Reaction buffer is composed of 0.10 M sodiumphosphate, 1.0 mM ethylenediaminetetraacetic acid, and 5.0 mM magnesiumchloride, pH 7.4 (all reagents: Fisher Scientific). Inhibitory chemicalsused were ketoconazole (Research Diagnostics. Inc.), miconazole,bergapten, xanthotoxin (previous three from Sigma), bergamotin,imperatorin, isopimpinellin, psoralen, angelicin (previous five fromIndofine Chemical Company, Inc.), and fractions or precipitatesresulting from Protocols A, A′, B, B′, or B″ above. When possible, finalinhibitor concentrations were expressed in molarity by calculation fromthe weighed material or by interpolation from HPLC calibration curvesprepared with reference materials; otherwise, concentrations areexpressed as weight per volume. Reaction tubes are placed on ice inpreparation for the manipulations that follow. Sufficient reactionbuffer is added so that the final volume of each tube will be 500 uL, 5uL of a 100-fold concentrate for generating reduced nicotinamide adeninedinucleotide phosphate is added (such that completed reaction mixturecontains 1.0 mM nicotinamide adenine dinucleotide phosphate, 1 U/mLglucose-6-phosphate dehydrogenase, and 10 mM glucose-6-phosphate: allfrom Sigma), and then human hepatic S9 (Anatomic Gift Foundation) isthawed and added in sufficient amounts to cause readily detectableamounts of metabolites to be formed in control reactions (amountnecessary varies among individuals, but 10 uL is typical). Reactions arepre-incubated for 3 min at 37° C. in a Dubnoff-type water bath, thereaction mixture is completed by the addition of 10 uL of 100 uMterfenadine (Sigma) dissolved in 1:1 acetonitrile:water and by gentlemixing, the samples are incubated for 15 min at 37° C., and the reactionis stopped by placing the tube on ice and adding 2.5 mL of 300 nMterfenadine-related compound A (internal standard; U.S. Pharmacopeia)dissolved in acetonitrile.

The samples prepared above are readied for HPLC assessment using theprotocol that follows. Each tube is vortex mixed and centrifuged for 10min at setting 35, the resulting supernatant is transferred to a cleantube, and the liquid is evaporated using a Speed Vac apparatus. Theresidue in each tube is first dissolved in 40 uL 1:1 acetonitrile:water,2.5 mL of acetonitrile is added, and the centrifuge-transfer-evaporatestep just described is repeated.

The dry residue resulting from the above-described experiments andsample preparation protocol may be analyzed for terfenadine metabolitesusing the HPLC method described below and may also be used to quantitatethe inhibitory chemicals that were added to the reaction (see ProtocolsB and B′). Linear gradients are used for elution and are formed bymixing mobile phase A composed of water with mobile phase B composed of0.025% (v/v) formic acid in acetonitrile (instrument: Hewlett Packard).The elution time, in minutes, the percentage of mobile phase B presentin the mixed mobile phase, and the flow rate (mL/min) are as follows: 0,10, 0.10; 2, 10, 0.10; 3.5, 10, 0.20; 4, 10, 0.25; 5, 10, 0.25; 30, 55,0.25; 32, 98, 0.25; 33, 98, 0.40; 39.8, 98, 0.40; 40, 98, 0.25; 45, 10,0.25; 45.25, 10, 0.20; 50, 10, 0.20; 50.25, 10, 0.10. Thechromatographic column has dimensions of 150 mm length×2.1 mm internaldiameter, is packed with a proprietary material (Prism, KeystoneScientific, Inc.), is protected with a 2 mm internal diameter columncontaining the same material and with a PTFE filter, and is maintainedat 35+/−0.2° C. The dry sample residue is mixed with 60 uL of 1:1acetonitrile:water followed by 40 uL water just prior to each 50.25 minrun cycle. The column eluate from each 10 uL injection is monitored forfluorescence with excitation at 228 nm, emission at 291 nm, and bandpassfiltration at 280 nm. Under these conditions, the retention times ofterfenadine alcohol metabolite, terfenadine carboxylic acid metabolite,and the internal standard are 16.2 min, 17.4 min, and 22.2 min,respectively.

Protocol C′: Assessment of Human Cytochrome P450-MediatedBiotransformation

The process of preparing incubation mixtures begins by mixing 10 uL ofethanol (control reactions) or an ethanolic solution containing aninhibitor with 100 uL of 100 mg/mL bovine serum albumin (Sigma)dissolved in reaction buffer at room temperature. Reaction buffer iscomposed of 0.10 M sodium phosphate, 1.0 mM ethylenediaminetetraaceticacid, and 5.0 mM magnesium chloride, pH 7.4 (all reagents: FisherScientific). Inhibitory chemicals used are ketoconazole (ResearchDiagnostics, Inc.), ritonavir (Norvir™, Abbott Laboratories), inhibitorychemicals described in Protocol C, and fractions resulting from ProtocolB″ above. Final inhibitor concentrations were expressed in molarity bycalculation from the weighed material or by use of Beer's law. Reactiontubes are placed on ice in preparation for the manipulations thatfollow. Sufficient reaction buffer is added so that the final volume ofeach tube will be 500 uL, 5 uL of a 100-fold concentrate for generatingreduced nicotinamide adenine dinucleotide phosphate is added (such thatcompleted reaction mixture contains 1.0 mM nicotinamide adeninedinucleotide phosphate, 1 U/mL glucose-6-phosphate dehydrogenase, and 10mM glucose-6-phosphate; all from Sigma), and then human hepatic S9(Anatomic Gift Foundation) is thawed and added in sufficient amounts tocause readily detectable amounts of metabolites to be formed in controlreactions (amount necessary varies among individuals, but 10 uL istypical). Reactions are pre-incubated for 3 min at 37° C. in aDubnoff-type water bath, the reaction mixture is completed by theaddition of 10 uL of 500 uM saquinavir (Invirase™, Roche Laboratories)dissolved in 1:1 ethanol:water and by gentle mixing, the samples areincubated for 15 min at 37° C., and the reaction is stopped by placingthe tube on ice and adding 2.5 mL of acetonitrile.

The samples prepared above are readied for HPLC assessment using theprotocol that follows. Each tube is vortex mixed and centrifuged for 10min at setting 35, the resulting-supernatant is transferred to a cleantube, and the liquid is evaporated using a Speed Vac apparatus. Theresidue in each tube is first dissolved in 40 uL 1:1 acetonitrile:water,2.5 mL of acetonitrile is added, and the centrifuge-transfer-evaporatestep just described is repeated.

The dry residue resulting from the above-described experiments andsample preparation protocol may be analyzed for saquinavir andsaquinavir metabolites using the HPLC method described below and mayalso be used to quantitate the inhibitory chemicals that were added tothe reaction (see Protocols B and B′). Linear gradients are used forelution and are formed by mixing mobile phase A composed of water withmobile phase B composed of acetonitrile (instrument: Hewlett Packard).The elution time, in minutes, and the percentage of mobile phase Bpresent in the mixed mobile phase are as follows: 0, 10; 5, 10; 30, 80;31, 95; 40, 95; 43, 10; 48, 10. The flow rate is 0.2 mL/min throughoutthe run. The chromatographic column has dimensions of 150 mm length×2.1mm internal diameter, is packed with a proprietary material (Prism.Keystone Scientific, Inc.), is protected with a 2 mm internal diametercolumn containing the same material and with a PTFE filter, and ismaintained at 35+/−0.2° C. In order to minimize the degradation ofanalytes, the dry sample residue is mixed with 50 uL of 1:1acetonitrile:water just prior to each 48 min run cycle. The columneluate from each 10 uL injection is monitored for absorbance at 239+/−2nm. Under these conditions, the retention times of saquinavir principalmetabolite A, saquinavir principal metabolite B, and saquinavir are 24.2min, 26.0 min, and 30.0 min, respectively.

Demonstration of Effectiveness

In order to demonstrate the first-pass effectiveness of the presentinvention, experiments with invention compounds and inventioncitrus-derived substances were conducted according to Protocol C′ abovewhere generation of saquinavir metabolites were measured in the presenceof various concentrations of inhibitor. Citrus-derived substancesaccording to the present invention were prepared according to ProtocolsA′, B′, and B″ above and were compared to known inhibitor Ketoconazole.FIG. 1 shows results for invention compounds of Formulae XI and XIII andalso shows that bergamottin and imperatorin are essentially ineffectivefirst-pass inhibitors. FIG. 2 shows how invention compounds compare toknown inhibitors Ritonavir and Ketoconazole.

In the invention compounds it is preferred that the furan ring positionof the furocoumarin rings be completely free of substitution.

With regard to purification and processing methods, the followingembodiments are preferred:

A. A method for processing citrus and selectively removing phototoxicfurocoumarins from a first-pass effective citrus-derived substance,comprising the step of extracting said citrus-derived substance with amixture of at least one C₂-C₄ alcohol, water, and optionally base, saidfirst-pass effective citrus-derived substance maintaininganti-first-pass activity after said extraction.

B. The method of embodiment A, wherein said citrus-derived substance isa cold-pressed citrus oil.

C. The method of embodiment A, wherein said mixture of ethanol and wateris a 30/70 volume/volume mixture of ethanol and water optionallycontaining 1-10% potassium hydroxide (W/V).

Other preferred embodiments include:

D. A first-pass effective citrus-derived substance which has beenextracted with a mixture of at least one C₂-C₄ alcohol and water so asto reduce the amount of phototoxic furocoumarins therein.

E. The citrus-derived substance of embodiment D, wherein said substanceis a cold-pressed citrus oil.

F. The citrus-derived substance of embodiment D, which has beenextracted with a 30/70 volume/volume mixture of ethanol and wateroptionally containing 1-10% potassium hydroxide (W/V).

G. A method for inhibiting the first-pass-effect of a material takenorally by a patient which is subject to the first-pass effect,comprising the step of co-administering to said patient the first-passeffective citrus-derived substance of embodiments A and D.

Invention compositions preferably comprise invention inhibitor material(compound, etc.) (e.g., alone, mixed with drug(s), and/or diluent(s)and/or carrier(s) etc.) such that they inhibit saquinavirbiotransformation according to Protocol C′ above better than pureketoconazole on an equal molar (preferred) or weight concentrationbasis. Alternatively, invention compounds, compositions, mixtures,formulations, etc. (materials) preferably provide a Y-axis value inProtocol C′ (see FIG. 1) of less than 0.5, preferably 0.45, 0.4, 0.35,0.3, 0.25, 0.22, 0.2, 0.18, 0.15, 0.12, 0.1, 0.08, 0.05 or 0.03 or lesswhen 0.01-0.25 mg, including 0.02, 0.04, 0.06, 0.08, 0.1, 0.12, 0.14,0.16, 0.18, 0.2, 0.22 and 0.24, and all ranges between all values, ofinvention material is diluted or dissolved to one liter.

A commercial form of citrus containing the invention compounds iscold-pressed grapefruit oil, which has a total concentration ofcompounds of Formulae XI-XVI in the range of perhaps up to 0.15-0.25mg/ml. The six compounds are distributed as: XI+XII+XIII+XIV equalsapproximately 50%, with XV and XVI the remainder. For XI-XIV thedistribution is about 3:2:2:1, respectively and about 2:1 for XV:XVI.Compositions according to the invention in one embodiment thuspreferably contain higher concentrations of invention compounds (i.e.,total concentration of all invention compounds therein) than those whichoccur in nature and commercial forms of citrus. These concentrations arereferred to as “concentrated amounts”. Examples of preferredconcentrations include greater than 0.25 mg/ml, 0.3, 0.8, 1, 2, 5, 8,32, 128, 200 mg/ml, etc. With regard to invention compounds, thecompounds in one embodiment of the invention are in a form distinct fromthat found in nature or commercially due to purity. The term“substantially pure” and “substantially pure form” refers to a puritygreater than that found commercially and in nature for the inventioncompounds. These concentrations and forms are easily derminable by thoseof ordinary skill now that the present inventor has identified theactive compounds responsible for the “grapefruit effect”. Otherlanguage, phrases, etc. useful to describe the embodiments of thepresent invention and distinguish them patentably and otherwise fromnaturally or commercially occurring forms are found in the followingpatents assigned to the U.S. government and to others, all incorporatedherein by reference: U.S. Pat. Nos. 4,708,948, 5,409,938, 5,455,251,4,977,244, 5,462,956, 5,314,899, 5,104,977, 5,484,889, 5,591,770,5,599,839, 5,672,607, 5,674,900, 5,648,354, 5,691,386, 5,681,829 and5,654,432. Another description of how the present invention may be used,in what amounts, and how administered appears in U.S. Pat. No.5,665,386, WO 97/15269 and WO 96/40192, all incorporated herein byreference.

The following patent applications, provisional or otherwise, areincorporated herein by reference: Ser. Nos. 60/056,382, 60/048,183,60/043,878,Ser. Nos. 08/764,081 and 08/673,800.

Other compounds useful herein are described by the following formulaewhere R, L, E and HAr are as described above. As with the abovecompounds, these compounds include all stereoisomers, E-Z isomers, etc.Where naturally or commercially occurring, these compounds arepreferably in the forms described above regarding purity, concentration,etc. These compounds may be optionally substituted as compounds ofFormulae I-XVI are.

In the invention compounds a preferred group of substituents, optionaland otherwise, comprise the following: hydrogen, C₁-C₄ alkyl, —S(C₁-C₄alkyl), —O(C₁-C₄ alkyl), —NH₂, —NH(C₁-C₄ alkyl)—N(C₁-C₂ alkyl) (C₁-C₄alkyl), hydroxy, —O(C₁-C₂ alkyl), fluoro, C₁-C₆ alkyl, chloro, bromo,iodo, C₁-C₄ alkoxy, —CF₃, —C(═O)O—(C₁-C₄) alkyl, —OC(═O)(C₁-C₄ alkyl),—OC(═O)N (C₁-C₄ alkyl) (C₁-C₂ alkyl), —NHCO(C₁-C₄ alkyl), —COOH,—COO(C₁-C₄ alkyl), —CONH(C₁-C₄ alkyl), —CON(C₁-C₄ alkyl) (C₁-C₂ alkyl),—S(C₁-C₄ alkyl), —CN, —NO₂, —SO(C₁-C₄ alkyl), —SO₂(C₁-C₄ alkyl),—SO₂NH(C₁-C₄ alkyl) and —SO₂N(C₁-C₄ alkyl) (C₁-C₂ alkyl).

Another group of preferred substituents, optional and otherwise,comprise: C₁-C₁₂ alkyl, aryl, (C₁-C₄ alkylene) aryl, phenyl, naphthyl,thienyl, benzothienyl, pyridyl, quinolyl, pyrazinyl, pyrimidinyl,imidazolyl, furanyl, benzofuranyl, benzothiazolyl, isothiazolyl,pyrazolyl, pyrrolyl, indolyl, pyrrolopyridyl, oxazolyl and benzoxazolyl,C₃-C₈ cycloalkyl or (C₁-C₆ alkylene) (C₃-C₈ cycloalkyl), C₁-C₄ alkyl,benzyl, C₁-C₄ alkanoyl, C₁-C₆ alkoxy, —OC(═O) (C₁-C₆ alkyl),—OC(═O)N(C₁-C₄ alkyl)(C₁-C₂ alkyl), —S(C₁-C₆ alkyl), amino, —NH(C₁-C₂alkyl), —N(C₁-C₂ alkyl) (C₁-C₄ alkyl), —N(C₁-C₄ alkyl)-CO—(C₁-C₄ alkyl),—NHCO(C₁-C₄ alkyl), —COOH, —COO(C₁-C₄ alkyl), —CONH(C₁-C₄ alkyl),—CON(C₁-C₄ alkyl) (C₁-C₂ alkyl), —SH, —SO(C₁-C₄ alkyl), —SO₂(C₁-C₄alkyl), —SO₂NH(C₁-C₄ alkyl) and —SO₂N(C₁-C₄ alkyl) (C₁-C₂ alkyl)

A third group of preferred substituents, optional and otherwise,comprise: —S(C₁-C₄ alkyl) or —SO₂(C₁-C₄ alkyl) (C₁-C₆ alkyl), —N(C₁-C₄alkyl) (C₁-C₂ alkyl), —S(C₁-C₄ alkyl), —SO(C₁-C₄ alkyl), —CO(C₁-C₄alkyl), —C(═O)H, —C(═O)O(C₁-C₄ alkyl), C₁-C₃ alkoxy, dimethylamino,methylamino, ethylamino, —NHC(═O)CH₃, C₁-C₃ thioalkyl, —COOH,—C(═O)O(C₁-C₄ alkyl), —C(═O)O(C₁-C₄ alkyl), —NO₂, phenyl, naphthyl,thienyl, benzothienyl, pyridyl, quinolyl, pyrazinyl, furanyl,benzofuranyl, benzothiazolyl, benzisothiazolyl, benzisoxazolyl,benzimidazolyl, indolyl, benzoxazolyl or C₃-C₈ cycloalkyl, chloro, C₁-C₆alkyl, —O(C₁-C₆ alkyl) bromo, iodo, formyl, —CN, —CF₃, —NO₂, —NH₂,—NH(C₁-C₄ alkyl), —N(C₁-C₂ alkyl) (C₁-C₆ alkyl), —C(═O)O(C₁-C₄ alkyl),—C(═O) (C₁-C₄ alkyl), —COOH, —SO₂NH(C₁-C₄ alkyl), —SO₂N(C₁-C₂ alkyl)(C₁-C₄ alkyl), —SO₂NH₂, —NHSO₂(C₁-C₄ alkyl), —S(C₁-C₆ alkyl) and—SO₂(C₁-C₆ alkyl), fluoro, hydroxy, amino, methylamino, dimethylamino,acetyl, hydrogen, C₁-C₄ alkyl, halo (e.g., chloro, fluoro, iodo orbromo), hydroxy, —O(C₁-C₄ alkyl), —C(═O) (C₁-C₄ alkyl), —C(═O)O(C₁-C₄alkyl), —OCF₃, —CF₃, —CH₂OH or —CH₂O(C₁-C₂ alkyl)hydroxy, methoxy andfluoro.

Within these three groups of preferred substituents are also specificexamples of HAr (i.e., C₆-C₂₄ aromatic groups or heteroaromatic groups).

In the present invention prodrugs and active metabolites of theinvention compounds, compositions, etc. are included. Such prodrugs arecompounds which give rise to an invention compound upon administrationto a mammal such as a human. Active metabolites are compounds formedupon administration of an invention compound, composition, etc. to amammal, preferably a human, which are first-pass effective. Someexamples of invention prodrugs and metabolites include:

Invention compounds, metabolites, prodrugs, etc. may preferably besubstituted with deuterium and/or fluorine to increase residence time inthe patient/animal/etc.

Pharmaceutical carriers, diluents, excipients, etc. are known to thoseof skill in this art. Examples are provided in several above-notedpatents and publications.

1. A substantially pure compound selected from the group consisting of:


2. The substantially pure compound of claim 1, of formula:


3. The substantially pure compound of claim 1 of formula:


4. A first-pass effective composition comprising at least one compoundselected from the group consisting of:

and a pharmaceutically acceptable carrier.